NZ330885A

NZ330885A - Process for preparation of vasoactive intestinal peptide analogs

Info

Publication number: NZ330885A
Application number: NZ330885A
Authority: NZ
Inventors: David Robert Bolin; Waleed Danho; Arthur M Felix
Original assignee: F
Priority date: 1996-02-09
Filing date: 1997-01-29
Publication date: 2000-02-28
Also published as: AU1596397A; CZ251698A3; WO1997029126A1; MX9806099A; BR9707409A; HUP9900961A3; KR19990082403A; HUP9900961A2; CN1210543A; ATE328004T1; NO983626L; CZ294779B6; EP0879246A1; DE69735991D1; US6080837A; JP2000504676A; AU722895B2; US6316593B1; CN1186355C; EP0879246B1

Abstract

This process is for the synthesis of vasoactive intestinal peptide analog Ac(VIP1-31)-NH2 from four protected peptide fragments: Leu Lys Lys Gly Gly Thr Ala Lys Lys Tyr Leu Asn Asp Asn Tyr Thr Leu Arg Lys Gln Xaa Ala His Ser Asp Ala Val Phe Thr Glu

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 330885 SYNTHESIS OF VIP ANALOG This invention relates to a novel process for the synthesis of vasoactive intestinal peptide analog Ac-(VIP1.3I)-NH2 fiom four protected peptide fragments Vasoactive intestinal peptide (VIP) is a smooth muscle relaxant/bronchodilator which regulates airway mucus secretion and has anti-allergic and antiinflammatory properties Recent studies have resulted in the discovery of an analog of VIP which possesses enhanced metabolic stability and has increased teceptor-biMi'iTg' properties This VIP analog is the subject of the European Patent Application No 92/117,185 To date, this VIP analog has been prepared using solid phase synthesis The solid phase synthesis includes attaching an alpha-amino acid, protecting with, for example t-butyloxycarbonyl (Boc), by ester linkage, to a chloromethylated resin or a hydroxymethyl resin More amino acids are added sequentially to the resin The alpha amino Boc protection is removed under acidic conditions and the subsequent protected ammo acids are coupled' stepwise to obtain an intermediate, protected peptide-resin Blocking groups are removed and the _ peptide is cleaved--from the resm through multiple hydrogen fluoride cleavage reactions -PuFi-fication -of- the -peptides occurs in two stages, a) size exclusion gel chromatography and b) preparative high performance liquid chromatography (HPLC) This multistep process is time consuming and results m inefficient recovery of the target peptide i intellectual property office 1 or nz 1 0 JAN 2000 s 10 - 2 - 33 o It is thus an object of the present invention to provide a relatively simple, more efficient and economic procedure for the synthesis of the VIP analog The present invention provides a novel process for the synthesis of a VIP analog, Ac-(VIP1.31)-NH2, having the formula 1 8 12 17 Ac-His-Ser-Asp-Ala-Val-Phe-Thr-Glu-Asn-Tyr-ThrT ys-Leu-Arg-Lys-Gln-Nle-Ala 19 25 26 27 28 29 30 31 Ala-Lys-Lys-Tyr-Leu-Asn-Asp-Leu-Lys-Lys-Gly-Gly-Thr-NH2 HN \ (SEQ ED NO 1) from protected peptide fragments Preferably, the reaction is characterized by the coupling of Fmoc-protected fragments, more preferably by the coupling of the following four fragments peptide Fragment I (SEQ ED NO 2), peptide Fragment II (SEQ ID 15 NO 3), peptide Fragment III (SEQ ED NO 4) and peptide Fragment IV (SEQ ID NO 5) The novel process does not require prior preparative HPLC purification of the peptide fragments as is required when the analog is prepared by solid phase synthesis, nor does it require purification of the intermediates formed 20 during the synthesis of the target cyclic VIP analog The resulting product was purified in the final stage after assembly by a single pass via preparative HPLC The method of the present invention for the synthesis of 25 the cyclic VIP analog Ac-(VIP,.31)-NH2 comprises the coupling of two or more protected peptide fragments Fieferred protected intellectual property office { of nz j 1 0 JAN 2000 f RECEIVED - 3 - fragments are the four Fmoc protected fragments Fragment I, Fmoc-(VIP26_3I)-NH2 (SEQ ID NO 2), Fragment II, Fmoc-(VIP19_25)-OH (SEQ ID NO 3), Fragment III, Fmoc-(VIP9.,8)~OH (SEQ ID NO 4), and Fragment IV, Ac-(VIP[ 8)-OH (SEQ ID NO 5), each of which is shown 5 below 26 31 -|-ys-Lp Boc Boc Fmoc-Leu-J-ys-Lys-Gly-Gly-Thr-NH2 tBu (I) Boc tBu 19 | | 25 Fmoc-Ala-Lys-Lys-Tyr-Leu-Asn-Asp-OH HN' V (II) 9 18 Fmoc-Asn-Tyr-Thr-Lys-Leu-Arg-Lys-Gln-Nle-Ala-OH M ! || tBu tBu Boc PmcBoc (HI) 1 8 Ac-His-Ser-Asp-Ala-Val-Phe-Thr-Glu-OH III II (IV) ^ Trt tBuOtBu tBuOtBu 10 The method of the present invention for the synthesis of a compound Ac-(VIP,_31)-NH2 (SEQ ID NO 1) by coupling four Fmoc protected peptide fragments, peptide Fragment I (SEQ ID NO 2), pepude Fragment II (SEQ ID NO 3), peptide Fragment III (SEQ ID NO IV) and peptide Fragment IV (SEQ ID NO 5) comprises 15 (a) deprotecting the Fmoc-protecting group of peptide Fragment I and coupling the deprotected peptide Fragment I with protected intellectual property office of nz I 0 JAN 2000 ! RECEIVED peptide Fragment II, (b) deprotecting the Fmoc-protecting group of the resulting peptide of step (a) and coupling it with protected Fragment III, (c) deprotecting the Fmoc-protecting group of the resulting peptide of step (b) and coupling it with protected Fragment IV, (d) deprotecting the resulting protected peptide of step (c) to yield deprotected Ac^VIP^^-NHt The protected peptide fragments I-IV were selected on the basis of maximum coupling efficiency and minimal racemization of the product of each coupling reaction Equivalent amounts of each fragment were used for each coupling reaction, providing an economic pathway to the target peptide The intermediates formed after each coupling were used directly for subsequent coupling reactions without further purification The purity of each fragment produced after solid phase synthesis, as described herein, was from about 82% to about 97% after a single purification step as determined by analytical HPLC, and each fragment was used for the synthesis of the cyclic VIP analog without further purification More particularly the method for the synthesis of the punfied compound of the formula Ac-(VIP|.31)-NH2 (SEQ ID NO 1) comprises (a) deprotecting the Fmoc-protecting group of peptide Fragment I (SEQ ID NO*2) and coupling the deprotected peptide Fragment I with protected peptide Fragment II (SEQ ED NO 3) yielding protected intermediate peptide Fmoc-(VIP19.31)-NH (SEQ ED NO 6), (b) deprotecting the Fmoc-protecting group of intermediate Fmoc-(VIP 19.3i)-NH2 and coupling the deprotected intermediate Fmoc-(VIP 19.3,)-NH2 with protected Fragment III (SEQ ID NO IV) yielding protected intermediate peptide Fmoc-(VIP9.3,) NH2 (SEQ ID NO 7), (c) deprotecting the Fmoc-protecting group of intermediate Fmoc -(VIP9_3i)-NH2 and coupling the deprotected intermediate Fmoc -(VIP9.3,)-NH2 with protected Fragment IV (SEQ ID NO 5) yielding protected intermediate peptide Ac-(VIP1.3[)-NH2 ;intellectual property office 1 ;of nz ;10 JAN 2000 ;RECEIVED ;25 ;30 ;- 5 - ;a (a) deprotecting the protected peptide Ac-(VIP|.3i)-NH2, and (e) ;purifying the deprotected peptide Ac-(VIPi-3I)~NH2, for example, via preparative HPLC ;5 As used herein, the nomenclature used to define the peptides is that typically used in the art, wherein the ammo group at the N-terminus appears to the left and the carboxyl group at the C-terminus appears to the right By natural ammo acids is meant one of the naturally occurring ammo acids found in ;10 proteins, i e , Gly, Ala, Val, Leu, He, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met, Phe, Tyr, Pro, Trp, and His Where the amino acid has isomeric forms it is the L form of the ammo acid that is represented, unless otherwise expressly indicated ;15 The following abbreviations or symbols are used to represent ammo acids in addition to those described elsewhere herein, protecting groups, solvents, reagents and the like ;Symbol Meaning ;20 Ac Acetyl ;Nle Norleucme ;Fra 9-Fluorenylmethyl ;DEPEA N,N-Dnsopropylethylamine ;DMF Dimethylformamide ;The suffixes "-OH" and "-NH2" following "VIP" refer to the free acid and amide forms of the polypeptide, respectively In the event neither suffix is used, the expression is intended to encompass both forms ;A cyclic peptide, as defined herein, is a peptide wherein the side chain carboxy terminus of one ammo acid in the peptide is attached covalently to the side chain amino terminus of another amino acid in the peptide chain via formation of an amide bond intellectual provtltlv ofrch OF NZ ;1 0 JAN 2000 i RECEIVED I ;6 w ^ j ;Several nomenclatures and symbols are utilized to represent a cyclic peptide The following are examples a cyclo(Lys21-Asp25)-Fmoc-Ala19.LyS(Boc)20-Lys21-Tyr(tBu)22_Leu23-A.sn24_Asp25_OH, ;b Fmoc-(VIP19_25)-OH, ;c Fmoc-[Alal9.A.sp25]_viP cyclo(21 ->25), ;d [Fmoc-(SEQ ID NO 3)-OH], ;e l;moc-[Alal9-Asp-^]-VIP cyclo (Lys^l —»Asp-^), ;f [Fmoc-(SEQ ED NO 3)-OH], ;a o ;Boc tBu 19 | j 25 ;Fmoc-Ala-Lys-Lys-T yr-Leu-Asn-Asp-OH ;HN- ;\ ;The above structures (a-g), and the representation using the "(SEQ ED NO )" each represent and define the same peptide having an amino acid sequence corresponding to a VIP peptide fragment in which an Fmoc group has been substituted for hydrogen at the N-termmus Additionally, an amide bond has been formed between the side chain carboxyl of the lysine at position 21 and the side chain amine of aspartic acid at position 25, thus forming the cyclic peptide fragment The above representations for the peptide structure are considered to be equivalent and interchangeable ;In the cyclic peptides of the present invention, the following configurations apply unless otherwise stated i intellectual property offiu \ OF NZ I ;1 0 JAN 2000 ;WO 97/29126 PCT/EP97/00380 ;- 7 - ;Ammo Acid Terminus of amino acid in chain bound to make cyclic peptide ;Lys e amino e ;Asp 6 carboxyl (C = beta) ;Glu 7 carboxyl (y = gamma) ;The peptide fragments which comprise the VIP analog of the present invention may be readily synthesized by any known conventional procedure for the formation of a peptide linkage 10 between amino acids Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha ammo group of an amino acid or residue thereof having its carboxyl group or other reactive groups protected and the free primar) carboxyl group of another amino 15 acid or residue thereof having its amino group or other reactive groups protected ;The process for synthesizing the peptide fragments comprising the VIP analog may be carried out by a procedure 20 whereby each amino acid in the desired sequence is added one at a time in succession to another amino acid or residue thereof or by a procedure whereby peptide fragments with the desired amino acid sequence are first synthesized conventionally and then condensed to provide the desired peptide ;25 ;Such conventional procedures for synthesizing the peptide fragments include, for example, any solid phase peptide synthesis method In such a method, the synthesis of the peptide fragments can be carried out by sequentially incorporating the 30 desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods [Merrifield, R B , J Amer Chem Soc 85, 2149-2154 (1963), Barany et al, The Peptides, Analysis, Synthesis and Biology, Vol 2, Gross, E and Meienhofer, J , Eds Academic Press 35 1-284 (1980)] ;Mimosa 15 04 51 ;WO 97/29126 PCT/EP97/00380 ;- 8 - ;Common to chemical syntheses of peptides is the protection of reactive siae chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical 5 reaction from occurring at that site until the protecting group is ultimately removed It is also well known to protect the alpha amino group on an amino acid or fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha amino protection group to allow a subsequent reaction 10 to take place at that site While specific protecting groups have been disclosed in regard to the solid phase synthesis method, it should be noted that each amino acid can be protected by a protective group conventionally used for the respective amino acid in solution phase synthesis ;15 ;Alpha amino groups may be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-20 mtrobenzyloxycarbonyl, p-bromo-benzyloxycarbonyl, p- ;biphenyl-isopropyloxycarbonyl, 9-fluorenyl-methyl-oxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz), aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), dnsopropylmethyloxycarbonyl, isopropyloxycarbonyl, and 25 allyloxycarbonyl Boc is most preferred for alpha amino protection ;Carboxyl groups may be protected by a suitable protecting group selected from aromatic esters such as benzyl (OBzl) or 30 benzyl substituted with lower alkyl, halo, nitro, thio, or substituted thio, i e , lower alkyl (1-7 carbon atoms), thio, ;aliphatic esters such as lower alkyl, t-butyl (Ot-Bu), cyclopentyl, cyclohexyl (OcHx), cycloheptyl, and 9-fluorenyImethyl (QFm) ;OBzl and OFm are most preferred for glumatic acid (Glu) OChx, 35 OBzl and OFm are most preferred for aspartic aid (Asp) ;Mimosa 15 04 51 ;WO 97/29126 ;- 9 - ;PCT/EP97/00380 ;Hydroxyl groups may be protected by a suitable protecting group selected from ethers such as benzyl (Bzl) or benzyl substituted with lower alkyl, halo, such as 2,6-dichlorobenzyi (DCB), mtro, or methoxy, t-butyl (t-Bu), tetrahydropyranyl, and triphenylmethyl (tntyl) Bzl is most preferred for serine (Ser) and threonine (Thr) Bzl and DCB are most preferred for tyrosine (Tyr) ;Side chain amino groups may be protected by a suitable protecting group selected from aromatic urethane-type protecting groups such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, 2-chlorobenzyl-oxycarbonyl, (2-C1-Z), p-nitro-benzyloxycarbonyl, p-bromo-benzyloxycarbonyl, p-biphenyl-isopropyl-oxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and p-methoxy-benzyloxy-carbonyl (Moz), aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), dnsopropylmethyloxycarbonyl, isopropyloxycarbonyl, and allyloxycarbonyl Z is most preferred for ornithine (Orn) 2-C1-Z and Fmoc are most preferred for lysine (Lys) ;Guamdmo groups may be protected by a suitable protecting group selected from nitro, p-toluenesulfonyl (Tos), Z, adamantyloxycarbonyl, and Boc Tos is most preferred for argimne (Arg) ;Side chain amide groups may be protected by xanthyl (Xan) No protection is preferred for asparagine (Asn) and glutamine (Gin) ;Imidazole groups may be protected by a suitable protecting group selected from p-toluenesulfonyl (Tos), 9-fluorenylmethyl-oxycarbonyl (Fmoc), tnphenylmethyl (tntyl), 2,4-dinitrophenyl ;Mimosa 15 04 51 ;WO 97/29126 ;- 10 - ;PCTTEP97/'J)380 ;(Dnp), Boc and benzyloxymethyl (Bom) Tos and Bom are most preferred for histidine (His) ;A protected ammo acid may be represented for purposes of 5 the present invention, for example, as Lys(Boc), Glu(OtBu), and Tyr(tBu) ;All solvents including methanol (MeOH), methylene chloride (CH2CI2), acetonitnle (CH3CN), ether, hexane and dimethyl-10 formamide (DMF) were purchased from Fisher or Burdick and Jackson Tnfluoroacetic acid (TFA) was purchased from Halocarbon and used without further purification Dnsopropyl-ethylamine (DIPEA), 1,2-ethanedithiol (EDT), dicyclohexyl-carbodnmide (DCC), N-hydroxy-succinimide (HOSu) and 15 thioanisole were purchased from Aldnch Chemical Co, Inc ;(Milwaukee, WI) 1-Hydroxybenzotnazole (HOBT) was purchased from Sigma Chemical Co (St Louis, MI), [2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) and benzotnazol-l-yloxy-tri-(dimethylamino)-phosphomum 20 hexafluorophosphate (BOP) were purchased from Richelieu ;Biotechnologies (St Hyacinthe, Quebec, Canada) -Methoxy-4-alkoxybenzyl alcohol copolystyrene 1% divinylbenzene (Sasrin-resin) was obtained from Bachem Bioscience Fmoc/tBu-protected amino acids were all of the L-configuration and were purchased 25 form Bachem, Inc (Torrance, CA) ;Analytical high performance liquid chromatography (HPLC) was earned out on an LDC Constrametric IIG equipped with a Gradient Master and spectromonitor HI UV-Vanable 30 Wavelength Detector and performed on a Lichrosorb RP-8 (5|i) column (4 6 mm x 25 cm), Eluant (A) 0 1M NaC104 (pH 2 5)-(B) CH3CN with a linear gradient in 20 minutes, Flow 1 ml/minute, Detection 214 nm HPLC of the protected intermediates, Fmoc-(19-31)-NH2 and Fmoc-(9-31)-NH2, was performed on a 35 Lichrosorb RP-8 (5fi) column (4 6 mm x 25 cm), cluant (A) ;Mimosa 15 04 51 ;WO 97/29126 ;- 11 - ;PCT/EP97/00380 ;NaC104 (pH 2 5) - (B) CH3CN, linear gradient 40-90% (B) in 20 minutes, flow 1 ml/minute, detection 214 nm HPLC of protected Ac-(1-31)-NH2 was carried out on the same column using (B) CH3CNIPrOH (1 1), linear gradient 60-95% (B) in 20 minutes 5 HPLC of the cyclic VIP analog was performed on (1) a Vydac C-18 column, eluant (A) H2O (0 1% TFA) - (B) CH3CN (0 1% TFA), linear gradient 15-30% (B) in 20 minutes, flow 1 ml/minute, detection ;214 nm (11) a Zorbax Protein Plus column, eluant (A) H2O (0 1% TFA)- (B) CH3CN (0 1% TFA), linear gradient 20-35% (B) in 20 ;10 minutes, flow 2ml/minute, detection 210 nm (111) a Lichrosorb RP-8 (5p.) columnm, eluant (A) NaC104 (pH 2 5) - (B) CH3CN, ;linear gradient 30% - 50% (B) in 20 minutes, flow 1 ml/minute, detection 206 nm Preparative HPLC was earned out on a Delta Prep 3000 system YMC ODS-A (120A, 15p.) column (4 7 x 50 cm), 15 eluant (A) H2O (0 1% TFA)- (B) CH3CN MeOH (1 1) (0 1% TFA), ;linear gradient 20-50% (B) in 3 h, flow 80 ml/minute, detection ;215 nm ;Fast atom bombardment mass spectra (FAB-MS) were 20 recorded on a Beckman VG2AB-1F or VG70E-HF mass spectrometer Amino acid analyses were performed on a Beckman Model 121M Amino Acid Analyzer The protected peptide fragment and free peptides were nydrolyzed in 6N HC1 (Pierce Chemical Co) in sealed, evacuated tubes at 110°C for 24 h ;25 ;In the present invention, the four peptide fragments I-IV were prepared via repetitive solid phase synthesis Coupling reactions throughout the syntheses were monitored by the Kaiser ninhydnn test to determine reaction progress and completion 30 (Kaiser et al, Anal Biochem , 34, 595-598 (1970) Preparation of the resins was monitored by UV analysis as follows ;The substitution of a protected amino acid (AA) resin (Fmoc-AA-resin) at any point in the synthesis procedure uses the 35 absorbance of N-(9-fluorenylmethyl) piperidine at 301 nm ;Mimosa 15 04 51 ;WO 97/29126 PCT/EP97/00380 ;- 12 - ;1 ;(e=7800) Between 4 to S mg of resin is accurately weighed in a test tube and treated with 0 5 ml of 20% pipendme in DMF For example, into a test tube containing 5 05 nig of Fmoc-Gly-resin, 0 5 ml 20% piperidine in DMF is added 0 5 ml 20% pipendine in 5 DMF in an empty test tube is used as a blank Over the next 15 minutes, the test tube with the Fmoc-Gly-resin is swirled two or three times to make sure all the resin has come into contact with the pipendine solution DMF is added to both tubes to bring the volume to 50 ml The spectrophotometer is zeroed at 301 nm J0 with the blank The absorbance of the Fmoc-substitution is calculated as follows ;A301 x Vol(ml) = 526 x (50) = 0 67 mmol/g ;7800 x wt (g) 7800 x 00505 (g) ;15 ;Generally, deprotection of the Fmoc protecting group from the peptide resin fragments was conducted acording to the following procedure ;20 ;Protocol 1 Fmoc-Deprotection ;Step ;Reagents ;Time ;1 ;CH2C12 ;2x3 ;2 ;DMF ;3 minutes ;3 ;25% Pipendine ;5 minutes ;4 ;25% Pipendine ;15 minutes ;5 ;DMF ;3 minutes ;6 ;MeOH ;3 minutes ;7 ;CH2CI2 ;3 minutes ;8 ;MeOH ;3 minutes ;9 ;CH2CI2 ;3 x 3minutes ;10 ;DMF ;3 minutes ;1 1 ;Coupling ;90 minutes ;Mimosa 15 04 51 ;- 13 - ;33 o ;Q { s ;0 ;1 2 ;DMF ;3 minutes ;1 3 ;MeOH ;3 minutes ;1 4 ;CH2CI2 ;3 minutes ;1 5 ;MeOH ;2x3 minutes ;The synthesis of Fragment I, Fmoc-(VIP26.31)-NH2, hereinafter Fmoc-(26-31)-NH2, descnbed in Examples 1 to 8, included preparing a C-terminus amide fragment using an XAL-linkei resin and using oenzoti lazol-1 -yloxyti ls-(dimethylamino) phosphonium hexafluorophosphate (BOP) as the coupling leagent Generally, double coupling was performed for each amino acid to ensuie final purity Typically, four equivalents of leagents weie used foi the first coupling and two equivalents of leagents were used foi the second coupling ;15 ;20 ;25 ;30 ;The synthesis of Fragment II, Fmoc-(VIP19.25)-OH, hereinafter Fmoc-(19-25)-OH, described in Examples 9 to 15, comprised a stepwise solid phase assemblage of the piotected heptapeptide m which the COOH-terminal lesidue Asp, was linked to Sasnn-iesin at the p-COOH via Fmoc-Asp(0-Sasnn-OBzl The starting resin was coupled with the dipeptide, Fmoc-Leu-Asn-OH which was prepared fiom Fmoc-Leu-OH and H-Asn-OH (via preactivaaori of Fmoc-Leu-OH to Fmoc-Leu-OSu) in 71 3% yield (estimated purity >95% by analytical HPLC) The tripeptide-resin was subjected to two cycles of solid phase synthesis with Fmoc-Tyr(tBu)-OH and Fmoc-Lys(Alloc)-OH, respectively A portion of the resultant pentapeptide-resm was subjected to two more cycles of solid phase synthesis, in turn, with Fmoc-Lys(Boc)-OH and Fraoc-Ala-OH An aliquot of the fully protected heptapeptide-resin was cleaved with 0 5% TFA-CH2CI2 and gave 1 major peak (estimated purity >82%) by HPLC Selective removal of the Lys(Alloc)-1-protecting group was achieved with Pd[(C6H5)3P]2 and Bu3SnH and the resultant partially protected hepcapeptide-resm was cleaved with 0 5% TFA-CH2CI2 A total of five 10-minute cleavages was required to completely cleave the resin The intellectual property office of nz ;1 0 JAN 2000 ;I RECEIVED ;- 14 ;product, Fmoc-Ala-Lys(Boc)-Lys-Tyr(tBu)-Leu-Asn-Asp(OBzl) was determined to be >80% pure by analytical HPLC and obtained in an overall yield of 75 7% (compared to the loading of the starting resin, Fmoc-Asp(O-Sasnn)-OBzl) ;5 ;The side-chain to side-chain cyclization (Lys-^ to Asp-^) of the linear heptapeptide was carried out m solution containing DMF using BOP and DIPEA and was complete in 1 25 hours Analytical HPLC of this crude product revealed nearly complete 10 conversion of the linear heptapeptide A single purification step of the resultant cyclic heptapeptide was earned out on silica gel This purification step removed any oligomers formed during the lactamization Analytical HPLC of the purified product revealed that the cyclic heptapeptide was >98% pure The overall yield of 15 the purified cyclic heptapeptide compared to the starting resin was 37% ;Final deprotection of the C-terminal benzyl ester was achieved by hydrogenolysis m a vibromixer apparatus using 10% 20 Pd on carbon over a period of about 6 hours The reaction was followed by analytical HPLC and the final product, cyclo(Lys21-Asp25)-Fmoc-Alal9-Lys(Boc)20-Lys-l- -Tyr(tBu)22_Leu-3- ;Asn-^-Asp^-OH was obtained in an overall yield of 33 7% (compared to the starting resin) with >97% purity The structure 25 and identity of the final product was confirmed by ^H-NMR ;spectroscopy and fully characterized by ammo acid analysis and 1 mass spectroscopy ;Repetitive solid phase synthesis of Fragment III, Fmoc -(VIP9.,8)-30 -OH, heieinaftei Fmoc-(9-l 8)-OH, described in Examples 16-26, and Fragment IV, Ac-(VIP,_s)-OH, heieinafter Ac-(l-8)-OH, ;described m Examples 27-36, was carried out using BOP as the coupling reagent and pipendine fot the deprotection of the Fmoc protecting group Highly acid-labile Sasrin linker was 35 used to retain side-chain piotecting groups on the fragments ;Generally, two coupling reactions were performed for each amino intellectual property office j of nz ! ;1 0 JAN 2000 ! ;I ;RECEIVED j ;WO 97/29126 ;- 15 - ;PCT/EP97/00380 ;acid to ensure final purity Two equivalents of reagent were used for the first coupling and one equivalent of reagent was used for the second or third coupling ;5 The average purity obtained after cleavage was 91% for peptide fragment III and 95% for peptide fragment IV These peptide purities obtained after cleavage were satisfactory for direct use m the peptide fragment covergent synthesis of the VIP analog without further purification ;0 ;The synthesis of the novel cyclic VIP analog of the present invention was achieved by the coupling of the four fragments I-IV and is illustrated below ;Mimosa 15 04 51 ;WO 97/29126 ;- 16 ;PCT/EP97/00380 ;H+ ;26 ;31 ;-bNH2 ;(I) ;19 21 25 Fmoc-| 1 1— OH ;hn' ;(H) ;Fmoc —1 —I- ;HBTU/HOBt ;19 21 25 ;HN V ;-i-nh2 ;Fmoc ;(in) ;18 ;H-oh ;ET2NH HBTU/HOBt ;Fmoc -1 ;21 ;HN" ;31 ;-I -NH2 ;Ac -I — ;(IV) ;-OH ;ET,NH HBTU/HOBt ;1 21 25 31 Ac_, ,-Wfc ;I TFA ;1 21 25 31 ;Ac-I ,'NHa deprotected ;Mimosa 15 04 51 ;WO 97/29126 PCT/EP97/00380 ;- 17 - ;Each cycle of fragment coupling was carried out by the same procedure as follows (1) deprotection of the Fmoc-protecting group of one peptide fragment with 10% Et2NH in DMF (2 hours), (u) removal of fluorene by washing with hexane-5 ether, (111) coupling of the deprotected peptide fragment with 1 0 equivalent of another protected fragment using HBTU (1 2 eq), HOBt (3 6 eq) in DMF-CH2CI2 at 0730 minutes-lh and 25°/3h using DIPEA (4 8 eq), and (iv) evaporation and dissolution in CH2CI2 and extraction with saturated NaHC03 and 10% citric acid ;10 ;In a preferred embodiment, each cycle of fragment coupling was carried out as follows (1) deprotection of the Fmoc-protecting group ot peptide Fragment I with 10% Et2NH in DMF (2 hours), (11) removal of fluorene by washing with hexane-ether, (111) 15 coupling of deprotected peptide Fragment I with 1 0 equivalent of protected peptide Fragment II using HBTU (1 2 eq), HOBt (3 6 eq) in DMF-CH2CI2 at 0730 minutes-lh and 2573h using DIPEA (4 8 eq), and (iv) evaporation and dissolution in CH2CI2 and extraction with saturated NaHC03 and 10% citric acid yielding 20 protected intermediate Fmoc(l9-31 )-NH2, (v) deprotection of the Fmoc-protecting group of intermediate Fmoc(19-31)-NH2 with 10% Et2NH in DMF (2 hours), (vi) removal of fluorene by washing with hexane-ether, (vn) coupling of deprotected intermediate Fmoc( 19-3 1 )-NH2 with 10 equivalent of protected Fragment III 25 using HBTU (1 2 eq), HOBt (3 6 eq) in DMF-CH2CI2 at 0730 minutes-lh and 2573h using DIPEA (4 8 eq), and (vm) ;evaporation and dissolution in CH2CI2 and extraction with saturated NaHC03 and 10% citric acid yielding protected intermediate Fmoc(9-31)-NH2, (ix) deprotection of the Fmoc-30 protecting group of intermediate Fmoc(9-31)-NH2 with 10% ;Et2NH in DMF (2 hours), (x) removal of fluorene by washing with hexane-ether, (xi) coupling of deprotected intermediate Fmoc(9-31)-NH2 with 10 equivalent of protected Fragment IV using HBTU (1 2 eq), HOBt (3 6 eq) in DMF-CH2CI2 at 0730 minutes-lh 35 and 2573h using DIPEA (4 8 eq), and (xn) evaporation and ;Mimosa 15 04 51 ;WO 97/29126 ;- 18 - ;PCT/EP97/00380 ;dissolution m CH2CI2 and extraction with saturated NaHC03 and 10% citric acid yielding protected intermediate Ac-(1-31)-NH2 ;The fragment convergent synthesis of the cychc VIP analog 5 is described in detail in Examples 37 through 41 The protected intermediates, Fmoc(19-31)-nh2, Fmoc(9-31)-nh2, and Ac(l-31)-nh2 were obtained as the major product from each reaction mixture cycle These crude protected intermediates were not purified further and were used in their crude forms for each 10 subsequent coupling cycle Analytical HPLC confirmed that the starting materials were fully consumed under the conditions employed ;Final deprotection of the fully protected peptide Ac(l-31)-15 NH2 was achieved by reaction with TFA (90%) EDT (3%), ;thioanisole (5%) and anisole (2%) at 25° for 2 hours Analytical HPLC confirmed that the cyclic VIP analog was the major product (72-75%) of the synthesis ;20 Purification of the crude product was achieved in a single pass by preparative HPLC using a YMC ODS-A reverse phase column (4 7 x 50 cm) Further scaling up to 4 0 g of crude product was readily achieved using this column and a total of 8 48 g of purified cyclic VIP analog, or 85 2% of the estimated 25 total of 9 95 g (from HPLC analysis) of cychc VIP analog present in the crude product, was isolated This corresponds to an overall yield of the synthesis of 23 9%, which includes the coupling reactions, deprotection and purification steps The cyclic VIP analog prepared by this method was shown to be homogeneous 30 (>99%) by analytical HPLC and capillary zone electrophoresis The identity of the cyclic VIP analog was confirmed by FAB mass spectroscopy and amino acid analysis ;Mimosa 15 04 51 ;WO 97/29126 ^ PCT/EP97/00380 ;EXAMPLES ;Examples 1-8 describe the solid phase synthesis of protected Fragment I, Fmoc-(26-31-NH2), Fmoc-Leu-Lys(Boc)-5 Lys(Boc)-Gly-Gly-Thr(t-Bu)-NH2 Examples 9 through 15 ;descnbe the synthesis of Fragment II, cyclo(Lys21-Asp25)_Fmoc-Alal9-Lys(Boc)20-Lys21- Tyr(tBu)22.Leu23-Asn24_Asp25.0H Examples 16 to 26 descnbe the solid phase synthesis of protected Fragment III, Fmoc-(9-18)-OH, Fmoc-Asn-Tyr(tBu)-Thr(tBu)-10 Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-OH Examples 27 to 36 describe the solid phase synthesis of Protected Fragment IV, Ac-(l-8)-OH, Ac-His(Trt)-Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-OH The synthesis of the cyclic VIP analog, Ac(l-31)-NH2, is described in Examples 37 to 41 ;15 ;EXAMPLE 1 Preparation of C3VXAL-Resin ;20 150g of benzhydrylamine (BHA) resin (loading 0 54 meq/g, ;lot # 13933) was neutralized with 2 x 1500 ml of NMP containing 10% triethylamine, then washed with 1000 ml of DMF, 1000 ml of MeOH, 1000 ml of CH2CI2, 1000 ml of MeOH and 3 x 1000 ml of CH2CI2 96 3 g of (3)-XAL-hnker (0 18 mol, 2 2 eq ) ,79 6 g of BOP 25 (0 18 mol) and 24 3 g of HOBt (0 18 mmol) were dissolved m 200 ml of NMP 47 03 ml of DIPEA was added and the solution was added in one portion to a reactor containing the neutralized resin The mixture was agitated for 90 minutes ;30 An aliquot was removed and washed with DMF and MeOH ;Loading of the XAL-lmker was determined by UV analysis to be 0 365 mmol/g The XAL-resin was washed with 1000 ml of DMF, 1000 ml of MeOH, 1000 ml of CH2CI2 and 2x 1000 ml of MeOH The uncoupled BHA resin was blocked with 1000 ml of 10% acetic 3 5 anhydride and 10% DIPEA in CH2CI2 for 30 minutes The resin ;Mimosa 15 04 51 ;WO 97/29126 ;- 20 - ;PCT/EP97/90380 ;was then filtered and washed with 1000 ml of CH2CI2, 1000 ml of MeOH, 2 x 1000 ml of CH2CI2 and 2 \ 1000 ml of MeOH ;EXAMPLE 2 ;5 ;Preparation of Fmoc-Thrft-BuVXAL-BHA ;For the first coupling, a mixture of 119 2 g of Fmoc-Thr(t-Bu)-OH (300 mmol), 132 g of BOP (300 mmol) and 40 5 g of HOBt 10 (300 mmol) was dissolved in 1000 ml of NMP with stirring at room temperature 78 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added m one portion to the XAL-resm prepared in Example 1 The mixture was agitated for 90 minutes ;15 ;After filtration, the resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;20 For the second coupling, a mixture of 59 6 g of Fmoc-Thr(t- ;Bu)-OH (150 mmol), 66 3 g of BOP (150 mmol) and 20 2 g of HOBt (150 mmol) were dissolved in 1000 ml of NMP with stirring at room temperature 39 2 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 25 reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes ;After filtration, the Fmoc-Thr(t-Bu)-XAL-BHA resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 30 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 ;- 21 - ;PCT/EP97/00380 ;EXAMPLE 3 Preparation of Fmoc-Glv-ThrCt-Bul-XAL-BHA ;5 Deprotection of the Fmoc protecting group of Fmoc-Thr(t- ;Bu)-XAL-BHA was conducted according to the procedure described in Protocol 1 Coupling of Fmoc-Gly-OH to Thr(t-Bu)-XAL-BHA was conducted as follows ;10 For the first coupling, a mixture of 89 2g of Fmoc-Gly-OH ;(300 mmol), 132 g of BOP (300 mmol) and 40 5 g of HOBt (300 mmol) was dissolved in 1000 ml of NMP with stirring at room temperature 78 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent 15 was added m one portion to the Thr(t-Bu)-XAL-BHA resin The mixture was agitated for 90 minutes ;After filtration, the resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 20 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;For the second coupling, a mixture of 44 6g of Fmoc-Gly-OH (150 mmol), 66 3 g of BOP (150 mmol) and 20 2 g of HOBt (150 mmol) were dissolved in 1000 ml of NMP with stirring at room 25 temperature 39 2 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes ;30 After filtration, the Fmoc-Gly-Thr(t-Bu)-XAL-BHA resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 ^ PCT/EP97/00380 ;EXAMPLE 4 ;Preparation of Fmoc-Glv-Glv-Thrft-BuVXAL-BHft ;5 Deprotection of the Fmoc protecting group of Fmoc-Gly- ;Thr(t-Bu)-XAL-BHA was conducted according to the procedure described in Protocol 1 Coupling of Fmoc-Gly-OH to Gly-Thr(t-Bu)-XAL-BHA was conducted as follows ;10 For the first coupling, a mixture of 89 2g of Fmoc-Gly-OH ;(300 mmol), 132 g of BOP (300 mmol) and 40 5 g of HOBt (300 mmol) was dissolved in 1000 ml of NMP with stirring at room temperature 78 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent 15 was added in one portion to the Gly-Thr(t-Bu)-XAL-BHA resin The mixture was agitated for 90 minutes ;After filtration, the resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 20 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;For the second coupling, a mixture of 44 6g of Fmoc-Gly-OH (150 mmol), 66 3 g of BOP (150 mmol) and 20 2 g of HOBt (150 mmol) were dissolved in 1000 ml of NMP with stirring at room 25 temperature 39 2 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes ;30 After filtration, the Fmoc-Gly-Gly-Thr(t-Bu)-XAL-BHA resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 ;- 23 - ;PCT/EP97/00380 ;EXAMPLE 5 ;Preparation of Fmoc-LysCBocVGlv-Glv-Thrft-But-XAL-BHA ;5 Deprotection of the Fmoc protecting group of Fmoc-Gly-Gly- ;Thr(t-Bu)-XAL-BHA was conducted according to the procedure described in Protocol 1 Coupling of Fmoc-Lys-(Boc)-OH to Gly-Gly-Thr(t-Bu)-XAL-BHA was conducted as follows ;10 For the first coupling, a mixture of 140 5g of Fmoc- ;Lys(Boc)-OH (300 mmol) , 132 g of BOP (300 mmol) and 40 5 g of HOBt (300 mmol) was dissolved in 1000 ml of NMP with stirring at room temperature 78 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 15 reagent was added in one portion to the Glv-Gly-Thr(t-Bu)-XAL-BHA resin The mixture was agitated for 90 minutes ;After filtration, the resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 20 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;For the second coupling a mixture of 70 3g of Fmoc-Lys(Boc)-OH (150 mmol), 66 3 g of BOP (150 mmol) and 20 2 g of HOBt (150 mmol) were dissolved in 1000 ml of NMP with stirring 25 at room temperature 39 2 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes ;30 After filtration, the Fmoc-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL- ;BHA resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 PCT/EP97/00380 ;- 24 - ;EXAMPLE 6 ;Preparation of Fmoc-Lvs(Boc)-LvsfBoc)-Glv-GIv-Thr(t-BnVXAT.- ;BHA ;5 ;Deprotection of the Fmoc protecting group of Fmoc-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA was conducted according to the procedure described in Protocol 1 Coupling of Fmoc-Lys-(Boc)-OH to Lys(Boc)-Gly-Gly-Thi(t-Bu)-XAL-BHA was conducted 10 as folio vs ;For the first coupling, a mixture of 140 5g of Fmoc-Lys(Boc)-OH (300 mmol), 132 g of BOP (300 mmol) and 40 5 g of HOBt (300 mmol) was dissolved in 1000 ml of NMP with stirring 15 at 100m temperature 78 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA resin The mixture was agitated for 90 minutes ;20 After filtration, the resin was washed with 1000 ml of DMF ;(3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;For the second coupling a mixture of 70 3g of Fmoc-25 Lys(Boc)-OH (150 mmol), 66 3 g of BOP (150 mmol) and 20 2 g of HOBt (150 mmol) were dissolved in 1000 ml of NMP with stirring at room temperature 39 2 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture 30 was agitated for 90 minutes ;After filtration, the Fmoc-Lys(Boc)-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 35 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 ;- 25 - ;PCT/EP97/00380 ;EXAMPLE 7 ;Preparation of Fmoc-Leu-Lvs(Boc)-Lvs(Boc)-5 Glv-Glv-ThrCt-BuVXAL-BHA ;Deprotection of the Fmoc protecting group was of Fmoc-Lys(Boc)-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA conducted according to the procedure described in Protocol 1 Coupling of 10 Fmoc-Leu-OH to Lys(Boc)-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA was conducted as follows ;For the first coupling, a mixture of 106g of Fmoc-Leu-OH (300 mmol), 132 g of BOP (300 mmol) and 40 5 g of HOBt (300 15 mmol) was dissolved in 1000 ml of NMP with stirring at room temperature 78 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added m one portion to the Lys(Boc)-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA resin The mixture was agitated for 90 minutes ;20 ;After filtration, the resin was washed with 1000 ml of DMF (3 minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;25 For the second coupling a mixture of 53g of Fmoc-Leu-OH ;(150 mmol), 66 3 g of BOP (150 mmol) and 20 2 g of HOBt (150 mmol) were dissolved in 1000 ml of NMP with stirring at room temperature 39 2 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent 30 was added in one portion to the resin, and the mixture was agitated for 90 minutes ;After filtration, the Fmoc-Leu-Lys(Boc)-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA resin was washed with 1000 ml of DMF (3 ;Mimosa 15 04 51 ;WO 97/29 L26 ;- 26 - ;PCT/EP97/00380 ;minutes), 1000 ml of MeOH (3 minutes), 1000 ml of CH2CI2 (3 minutes) and 2 x 1000 ml of MeOH (3 minutes) ;The final weight of the peptide resin fragment Fmoc-Leu-5 Lys(Boc)-Lys(Boc)-Gly-Gly-Thr(t-Bu)-XAL-BHA, also known as Fmoc-(26-31)-XAL-BHA, was 267g ;EXAMPLE 8 ;10 Cleavage of the Fmoc-C26-31)-XAL-BHA ;In a 500 ml round bottom flask lOg of the protected Fmoc-(26-31)-XAL-BHA was charged with 200 ml of 0 5% TFA in CH2CI2 The slurry was stirred for 0 5 minutes at room 15 temperature and then filtered The filttate was immediately adjusted to pH 7 by the addition of pyridine The filtrate was evaporated at room temperature on a rotovac The residue was triturated with 200 ml of distilled water then washed with 2 x 200 ml ot ether The resulting solid material was dried in vacuo 20 to give the protected Fmoc-(26-31)-NH2 ;The filtered peptide-resin was then treated five more times with 200 ml of 0 5% TFA solution for 15 minutes, followed by adjusting the pH to 7 with pyridine After evaporation, 25 trituration and drying, a sample from each of the cleavages was analyzed by HPLC The HPLC conditions were column Lichrosorb RP-18, 5 m, 25 cm, eluants (a) 0 1 M HCIO4/H2O (pH2 5), (b) MeCN, gradient 34% to 39% MeCN/20 minutes, flow rate 1 ml/minute, and detector 210 nm Knau ;30 ;All the peptide fragments above 90% purity were combined to give a total of 3 01 g The purity of the combined material was 94 5% ;Mimosa 15 04 51 ;WO 97/29126 ;- 27 - ;PCT/EP97/00380 ;EXAMPLE 9 Preparation of Fmoc-Asp(Q-Sasrin)-OBzl ;5 2-Methoxy-4-alkoxybenzyl alcohol copolystyrene 1% ;divinylbenzene cross-linked resin (Sasrin-resm, 25 g, 0 96 meq/g, 24 meq ) was washed with DMF (2 x 500 ml) and CH2C12 (3 x 500 ml) Fmoc-Asp-<>c-O-benzyl ester (35 g, 78 56 mmole, 3 27 eq ) in CH2CI2 (400 ml) was added and shaken using a 10 mechanical shaker A solution of DCC (16 2 g, 78 5 mmole, 3 27 eq) in CH2C12 (100 ml) was added followed by the addition of N-methylmorphohne (3 36 ml, 30 mmole, 3 27 eq ) and 4-dimethylaminopyridine (0 293 g, 2 4 mmole, 0 1 eq ) and shaken for 5 hours An aliquot was removed, washed with methanol and 15 dried The loading by UV analysis was determined to be 0 60 ;mmol/g-resm The resin was washed wth DMF (2 x 500 ml) and methanol (5 x 500 ml) and dried in vacuo to give 32 8 g of Fmoc-Asp(0-Sasnn-Resin)-0Bzl A 10 g portion of this resin was suspended in DMF (180 ml) and a solution of benzoic anhydride 20 (6 78g 30 mmol, 5 eq ) in DMF (40 ml) was added followed by DIPEA (5 22 ml, 30 mmole, 5 eq ) and the suspension shaken for 30 minutes The resin was washed with DMF (5 x 200 ml) and the substitution was determined to be 0 60 rnmol/g ;25 EXAMPLE 10 ;Synthesis of Na-Fmoc-L-Leucvl-L-Asparagine(Fmoc-Leu-Asn-OH>) ;Fmoc-L-Leu-OH (10 g,28 32 mmol) was dissolved in a 30 mixture of DMF (10 mI)-CH2Cl2 (70 ml) and cooled in an ice- ;bath N-Hydroxysuccinimide (3 58g, 31 14 mmol, 1 1 equiv ) and dicyclohexylcarbodnmnde (6 06 g, 3114 mmol, 1 1 equiv ) were added and the mixture was stirred at 0°C for 1 hour and 25°C for 14 hour The reaction mixture was cooled in an ice-bath, filtered 35 and the precipitate washed with CH2CI2 (40 ml) The filtrate was ;Mimosa 15 04 51 ;WO 97/29126 PCT/EP97/00380 ;- 28 - ;evaporated to dryness and the residue dissolved in a mixture of dioxane (80 ml)-H20 (10 ml) to form a solution of Fmoc-Leu-OSu A solution of anhydrous L-asparagine (5 606 g, 42 48 mmol, 1 5 equiv ) in 60 ml of Na2CC>3 (2 50 g, 42 28 mmol, 1 5 equiv ) was 5 added to the above solution of Fmoc-Leu-OSu An additional 10 ml of dioxane was added and the reaction mixture was stirred at 25°C for 2 hours Ethyl acetate (200 ml) was added to the reaction mixture and the pH adjusted to about 2 by the addition of 5% aqueous HC1 with snmng The EtOAc layer was separated and 10 the aqueous layer was extracted 4 more times with 100 ml each The combined EtOAc extract was washed with saturated NaCl (100 ml), H20 (3 x 100 ml), dned over anhydrous Na2S04, ;filtered and evporated to dryness The residue was dissolved in DMF (60 ml) with warming (40-45°C) and H2O added to the cloud 15 point The product crystallized after standing overnight It was filtered, washed with ether and recrystallized from DMF-H2O Lyophihzation from dioxane gave 9 45 g (71 3%) of product NMR and FAB mass spectroscopy confirmed the molecular formula C25H29N3O6 with an observed mass for (M+H)+, 468 3 The 20 product Fmoc-Leu-Asn-OH was shown to be >95% pure by analytical HPLC (Fig 4) HPLC conditions were column Lichrosorb RP-8 (5(0.), eluant (A) 0 IM NaC104 (pH 2 5) - (B) CH3CN, gradient 40%-80% (B) in 20 minutes, flow 1 ml/1 minute , ana detection 210 nm ;25 ;EXAMPLE] ;Preparation of Fmoc-Ala-Lvs(Boc)-Lvs( Alloc v Tvr(tBu)-Leu-Asn-Asp(Q-Sasnn)-OBzl ;30 ;Solid phase peptide synthesis was carried out as follows (20 ml of solvent/g of resin was used) 1) 20% piperidine/DMF, 1 minute, 2) 20 % Pipendine DMF, 10 minutes, 3) DMF, 4x2 minutes, 4) NMP, 1x2 minutes, 5) coupling of the protected 35 amino acids as described herein, and 6) DMF, 3x2 minutes ;Mimosa 15 04 51 ;WO 97/29126 PCT/EF97/00380 ;- 29 - ;Solid phase peptide synthesis was carried out as described above starting with 10 g, 0 60 mmol/g 6 0 mmol of Fmoc-Asp(0-Sasnn)-OBzl Fmoc-Leu-Asn-Asp(0-Sasrm-resin)-OBzl was 5 prepared by coupling the dipeptide, Fmoc-Leu-Asn-OH to H-Asp(Q-Sasrin-resin)-OBzl as follows ;To Fmoc-Asn-Leu-OH, 4 2 g, 9 mmol, 1 5 eq was added HBTU, 3 41 g, 9 mmol, 1 5 eq, HOBT, 4 13 g, 27 mmol, 4 5 eq, 10 DIPEA, 3 135 ml, 18 mmol, 3 eq, and NMP/CH2Cl2 (1 1) 220 ml The mixture was coupled for 1 25 hours For this particular coupling, Fmoc-Leu-Asn-OH was dissolved in 160 ml of NMP-CH2CI2 (1 1) Since mixing of NMP and CH2CI2 was exothermic the solvent was cooled prior to addition to the peptide and added 15 to the H-Asp(0-Sasrin-resin)-0Bzl followed by DIPEA and HOBt and the reaction vessel was shaken for 2 minutes Then 30 ml of cold CH2CI2 was added and finally a solution of HBTU dissolved in 20 ml of NMP-CH2CI2 (1 1) and 10 ml of NMP was added The ratio of NMP CH2C12 was kept (1 1) The pH of the coupling 20 reaction was maintained at 5-6 ;Fmoc-Tyr(tBu)-Leu-Asn-Asp(0-Sasnn-resin)-OBzl was prepared by coupling Fmoc-Tyr(tBu)-OH to the resin as follows to Fmoc-Tyr(tBu)-OH, 5 4 g, 12 mmol, 2 eq was added HBTU, 3 4 25 g, 12 mmol, 2 eq, HOBt, 1 8 g, 12 mmol, 2 eq, DIPEA, 5 75 ml, 33 mmol, 5 5 eq, and NMP, 220 ml This mixture was added to the resin and coupled for 1 hour ;Fmoc-Lys(Alloc)-Tyr(tBu)-Leu-Asn-Asp(0-Sasrin-resin)-30 OBzl was prepared by coupling Fmoc-Lys(Alloc)-OH to the resin as follows to Fmoc-Lys(AUoc)-OH, 5 4g, 12 mmol, 2 eq was added HBTU, 3 4 g, 12 mmol, 2 eq, HOBt, 1 8 g, 12 mmol, 2 eq, DIPEA, 5 75 ml, 33 mmol, 5 5 eq, and NMP, 220 ml This mixture was added to the resin and coupled for 1 hour ;35 ;Mimosa 15 04 51 ;WO 97/29126 PCT/EP97/00380 ;- 30 - ;After the addition of Fmoc-Lys(Alloc)21-OH, the resin was washed with methanol and dried to yield 13 3 g (0 45 mmol/g, ;5 98 mmol) of the protected Fmoc-(21-25)-(0-Sasnn-resin)-OBzl One-half of this peptide-resin (6 65 g, 2 99 mmol) was subjected ;5 to two additional cycles of solid phase synthesis as described above and coupled with Fmoc-Lys(Boc)-OH as follows to Fmoc-Lys(Boc)-OH, 2 8 g, 6 mmol, 2 eq was added BOP, 2 65 g, 6 mmol, 2 eq, HOBt, 0 918 g, 6 mmol, 2 eq, DIPEA, 3 25 ml, 18 65 mmol, ;6 2 eq, and NMP 120 ml This was added to the resin and coupled 10 for 1 hour, and Fmoc-Ala-OH, as follows to Fmoc-Ala-OH, 1 86 g, ;6 mmol, 2 eq was added HBTU, 2 27 g, 6 mmol, 2 eq, HOBt, 0 90 g, 6 mmol, 2 eq, and NMP 120 ml The mixture was added to the resin and coupled for 1 hour ;15 The resin was washed with DMF (3 x 120 ml) and methanol ;(4 x 120 ml) and dried in vacuo to give 7 3 g (0 39 mmol/g, 2 84 mmole) of protected Fmoc (19-25)-(0-Sasnn-resin)-0Bzl A portion of Fmoc (19-25)-(0-Sasrin-resin)-0Bzl was cleaved with 0 5% TFA-CH^C^ and evaluated by analytical HPLC to be >82% 20 pure ;EXAMPLE 12 ;Fmoc-Ala-Lvs(Boc)-Lvs-TvrftBu)-Leu-Asn-AspfO-Sasrin') 25 -OBzl via deprotection of Fmor-Ala-LvsfBoc)-LvsfAlloc)- ;Tvr(tBu)-Leu-Asn-Asp(Q-Sasnn)-OBzl ;The protected hexapeptide-resin Fmoc-Ala-Lys(Boc)-Lys(Alloc)-Tyr(tBu)-Leu-Asn-Asp(0-Sasrin)-OBzl (7 2 g, 0 39 30 mmol/g, 2 8 mmol) was suspended in 130 ml of CH2CI2 and bubbled with helium Acetic acid (0 330 ml, 5 75 mmol), bis(triphenyl phosphine) palladium dichlonde (0 138 g,0 196 mmol) and tributyltm hydride (3 165 ml, 119 mmol) were added and the peptide-resin was bubbled with helium for 1 5 hours and ;Mimosa 15 04 51 ;WO 97/29126 PCTYEP97/00380 ;- 31 - ;the reaction vessel was drained The above reaction was repeated a total of 5 times to ensure complete deprotection of the Alloc-group An aliquot of peptide-resin was cleaved with 0 5% TFA/CH2CI2 and analytical HPLC showed complete deprotection 5 of the Alloc group The resin was washed with CH2CI2 (2 x 120 ml) and methanol (4 x 120 ml) and dried in vacuo to give 7 1 g (0 40 mmol/g, 2 84 mmole) of the partially protected peptide-resin Fmoc-Ala-Lys(B oc)-Ly s-Tyr( tBu)-Leu-Asn-Asp(0-Sasrin)-OBzl ;10 ;EXAMPLE 13 ;Cleavage of Fmoc-Ala-LvsfBocVLvs-TvrftBuVLeu-Asn-AspfQ-Sasnn)-OBzl Preparation of Fmoc-Ala-15 LvsfBoOLvs-TvrftBuVLeu-Asn-Asp-OBzl ;7 1 g (2 84 mmol) of the partially protected Fmoc-( 19-25)-Sasnn-resin-OBzl was treated with 0 5% TFA m CH2CI2 (140 ml) for 10 minutes at room temperature, filtered and the filtrate 20 immediately adjusted to pH 6-7 with the addition of pyridine The peptide-resin was subjected to four more treatments with 0 5% TFA in CH2CI2 as described above and the filtrates were combined and evaporated The peptide was triturated with water, filtered, washed liberally with water and dried in vacuo 25 The crude peptide was washed with anhydrous ether and dried in vacuo to give 3 26 g (overall yield, 75 7%) of the partially protected Fmoc-Ala-Ly s(B oc)-Lys-Tyr( tBu)-Leu-Asn-Asp-OB zl which was determined to be >80% pure by analytical HPLC ;Mimosa 15 04 51 ;WO 97/29126 ;- 32 - ;PCT/EP97/00380 ;EXAMPLE 14 ;Cyclization of Linear Heptapeptide Preparation of ;Fmoc-Ala-Lvs(Boc)-Lvs-Tvr(tBu)-Leu-Asn-Asp-OBzl ;5 cvclo(lvs21 -»Asp25) ;A solution of the partially protected linear heptapeptide Fmoc-Ala-Lys(Boc)-Lys-Tyr(tBu)-Leu-Asn-Asp-OBzl 3 26 g (2 271 mmol) in 100 ml of DMF was added slowly over a period 10 of 20 minutes to a magnetically stirred solution of BOP reagent ;2 0 g (4 53 mmol, 2 eq ) and DIPEA 3 16 ml (18 14 mmol, 8 eq ) in 500 ml of DMF After stirring at room temperature for 1 25 hours, an aliquot was removed and analytical HPLC indicated that the linear peptide was completely cyclized The reaction mixture 15 was stirred for an additional hour, acidified with acetic acid and evaporated to about 20 ml and distilled water (200 ml) was added The precipitate was collected by filtration, washed thoroughly with distilled water and dried in vacuo The product was washed with anhydrous ether and dried to give 3 0 g of the 20 protected cyclic heptapeptide Analytical HPLC showed nearly complete conversion of the linear peptide to the cyclic heptapeptide This material was dissolved in 100 ml of 10% methanol in CH2CI2 The insolubles (oligomers) were removed by filtering the solution through celite and the filtrate evaporated to 25 give 2 85 g of crude protected cyclic peptide This material was dissolved m 100 ml of CH2CI2 and loaded onto a Waters Prep Pack Silica Gel Column (4 7 x 30 cm, 15-20 |i), flow rate 50 ml/minute, detection 280 nm The column was eluted in turn with CH2CI2 (500 ml), 5% MeOH in CH2C 12(2000 ml) and finally 30 eluted with 8% MeOH in CH2C12 The fractions containing pure peptide (as determined by analytic HPLC) were pooled, ;evaporated to dryness and lyophilized from dioxane to give 1 445 g of purified cyclic heptapepude (overall yield, 37%) which analytical HPLC revealed to be >98% pure ;35 ;Mimosa 15 04 51 ;WO 97/29116 ;- 33 - ;PCT/EP97/00380 ;EXAMPLE 15 ;Preparation of Fmoc-Ala-Lvs(Boc)-Lys-Tyr(tBu)-Leu-Asn-Asp-OH ;cvclonvs21-»Asp25) ;The fully protected cychc heptapeptide of Example 14 (1 445 g, 1111 mmol) was dissolved m 60 ml of MeOH m a vibromixer flask under a stream of helium To this was added 0 314 g of 10% Pd on carbon and the reaction mixture was hydrogenated for 3 5 hours in the vibromixer apparatus Analytical HPLC indicated that about 30% of the benzyl ester fully protected cyclic heptapeptide remained An additional 0 2 g of 10% Pd on carbon was added and hydrogenation was continued for 2 75 hours Analytical HPLC indicated that complete deprotection of the benzyl group had occurred The reaction mixture was filtered through celite and washed with MeOH The filtrate and washings were evaporated and lyophihzed from dioxane to give 1 225 g (91 1% yield, overall yield 33 7%) of cychc heptapeptide acid Analytical HPLC revealed that the product was >91% pure The product identity was characterized and confirmed by amino acid analysis, FAB-MS, optical rotation and 1H-NMR spectroscopy ;EXAMPLE 16 Preparation of Fmoc-Ala-Sasrin Resin ;50 g of 2-Methoxy-4-alkoxybenzyl alcohol copolystyrene 1% divinylbenzene cross-linked resin (Sasnn resin) was washed with 500 ml of methylene chloride, and 2 x 500 ml of DMF ;74 6 g of Fmoc-Ala-OH (240 mmol) was dissolved in 650 ml of CH2CI2/DMF (2 I volume ratio) The solution was cooled in an ice bath and 49 5 g of DCC (240 mmol) was added, followed by ;Mimosa 15 04 51 ;WO 97/29126 ;- 34 - ;PCT/EP97/00380 ;the addition of 1 46 g of 4-dimethylamino pyridine (12 mmol) ;and 6 27 ml of N-methylmorpholine (60 mmol) The mixture was agitated on an orbital rotary shaker for 9 hours ;5 The resin was filtered and washed with 500 ml of DMF (3 ;minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 500 ml of MeOH (3 minutes) ;An aliquot was removed, dried and the loading was 10 determined by UV qualitative analysis to be 0 54 mmol/g ;The resin was then washed with 2 x 500 ml of CH2CI2 (3 minutes) and 500 ml of DMF (3 minutes) The resin was then suspended in 400 ml of DMF, and 54 3 g of benzoic anhydride 15 (240 mmol) was added, followed by the addition of 100 ml of DMF, and 41 7 ml of dnsopropylethylamine (240 mmol) The suspension was shaken for 30 minutes The resin was filtered and washed with 500 ml of CH2CI2 (3 minutes), 500 ml of MeOH (3 minutes) 2 x 500 ml of CH2CI2 (3 minutes), 500 ml of DMF (3 20 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 17 Preparation of Fmoc-Nle-Ala-Sasnn Resin ;25 ;Deprotection of the Fmoc group of Fmoc-Ala-Sasnn Resin was conducted according to the procedure described in Protocol 1 The coupling of Fmoc-Nle-OH to Ala-Sasnn was conducted as follows ;30 ;For the first coupling a mixture of 19 08 g of Fmoc-Nle-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 14 1 ml of DIPEA was added to the above solution, ;Mimosa 15 04 51 ;WO 97/29126 ;- 35 - ;PCT/EP97/00380 ;and the mixture was vigorously stirred The resulting reagent was added in one portion to the Ala-Sasnn resin, and the mixture was agitated for 2 hours ;5 After filtration, the resin was washed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;For the second coupling, a mixture of 9 54 g of Fmoc-Nle-OH 10 (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml of NMP and stirred at 100m temperature 7 05 ml of DIPEA was added to the <ibove solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin and the mixture was 15 agitated for 2 hours ;After filtration, the Fmoc-Nle-Ala-Sasrin resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 20 minutes) ;EXAMPLE 18 Preparation of Fmoc-Gln-Nle-Ala-Sasnn Resin ;25 ;Deprotection of the Fmoc group of Fmoc-Nle-Ala-Sasrin was conducted according to the procedure described in Protocol 1 ;For the first coupling a mixture of 19 9 g of Fmoc-Gln-OH 30 (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 14 1 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Nle-Ala-Sasnn resin, and the 35 mixture was agitated for 2 hours ;Mimosa 15 04 51 ;WO 97/29126 ;- 36 - ;PCT/EP97/00380 ;After filtration, the resin was washed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 ;minutes) and 2 x 500 ml of MeOH (3 minutes) ;5 ;For the second coupling, a mixture of 9 9 g of Fmoc-Gln-OH (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml of NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, 10 and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc-Gln-Nle-Ala-Sasrin resin was 15 washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 ;minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;20 ;25 ;EXAMPLE 19 ;Preparation of Fmoc-LvsfBocl-Gln-Nle-Ala-Sasnn Resin ;Deprotection of the Fmoc group of Fmoc-Gln-Nle-Ala-Sasnn was conducted according to the piocedure described in Protocol 1 ;For the first coupling a mixture of 25 3 g of Fmoc-Lys(Boc)-Gln-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 14 1 ml of DIPEA was added to the above solution, 30 and the mixture was vigorously stirred The resulting reagent was added in one portion to the Gln-Nle-Ala-Sasrin resin, and the mixture was agitated for 2 hours ;Mimosa 15 04 51 ;WO 97/29126 ;- 37 - ;PCT/EP97/00380 ;After filtration, the resin was v/ashed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 ;minutes) and 2 x 500 ml of MeOH (3 minutes) ;5 For the second coupling, a mixture of 12 7 g of Fmoc- ;Lys(Boc)-OH (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml of NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 10 reagent was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc-Lys(Boc)-Gln-Nle-Ala-Sasnn resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 15 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 20 ;20 Preparation of Fmoc-Arg(Pmc)-Lvs(Boc)-GIn-Nle-Ala-Sasrin Re^in ;Deprotection of the Fmoc group of Fmoc-Lys(Boc)-Gln-Nle-Ala-Sasrin was conducted according to the procedure described in Protocol 1 ;25 ;For the first coupling a mixture of 40 1 g of Fmoc-Arg(Pmc)-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 14 1 ml of DIPEA was added to the above solution, ;30 and the mixture was vigorously stirred The resulting reagent was added in one portion to the Lys(Boc)-Gln-Nle-Ala-Sasnn resin, and the mixture was agitated for 2 hours ;Mimosa 15 04 51 ;WO 97/29126 ;- 38 - ;PCT/EP97/00380 ;After filtration, the resin was washed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 ;mmutes) and 2 x 500 ml of MeOH (3 minutes) ;5 For the second coupling, a mixture of 20 1 g of Fmoc- ;Arg(Pmc)-OH (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml of NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 10 reagent was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasnn resin was washed with 500 ml of DMF (3 minutes), 500 ml 15 of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 21 ;20 Preparation of Fmoc-Leu-ArgfPmc)-Lvs(Boc)- ;Gln-Nle-Ala-Sasrin Resin ;Deprotection of the Fmoc group of Fmoc-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasrin was conducted according to the 25 procedure described in Protocol 1 ;For the first coupling a mixture of 19 1 g of Fmoc-Leu-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room 30 temperature 14 1 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasnn resin, and the mixture was agitated for 2 hours ;Mimosa 15 04 51 ;WO 97/29126 ^9 PCT/EP97/00380 ;After filtration, the resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;5 For the second coupling, a mixture of 9 6 g of Fmoc-Leu-OH ;(27 mmol), 119 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml ol NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent 10 was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasrm resin was washed with 500 ml of DMF (3 minutes), 15 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 22 ;20 Preparation of Fmoc-LvsfBoc)-Leu-Arg(Pmc)- ;Lvs(Boc)-Gln-Nle-Ala-Sasrin Resin ;Deprotection of the Fmoc group of Fmoc-Leu Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasnn was conducted according to the 25 procedure described m Protocol 1 ;For the first coupling a mixture of 25 4 g of Fmoc-Lys(Boc)-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room 30 temperature 14 1 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Leu-Arg(Pmc)-Lys(Boc)-GIn-NIe-Ala-Sasrin resin, and the mixture was agitated for 2 hours ;Mimosa 15 04 51 ;WO 97/29126 ;- 40 - ;PCT/EP97/00380 ;After filtration, the resin was washed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 ;minutes) and 2 x 500 ml of MeOH (3 minutes) ;5 For the second coupling, a mixture of 12 7 g of Fmoc- ;Lys(Boc)-OH (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml of NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 10 reagent was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-AIa-Sasnn resin was washed with 500 ml of DMF (3 i5 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 23 ;20 Preparation of Fmoc-Thr(tBu)-LvsfBoc)-Leu-Arg«'Pmc)- ;LvsfBoc)Gln-Nle-Ala-Sasnn Resin ;Deprotection of the Fmoc group of Fmoc-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasrin was conducted according 25 to the procedure described in Protocol 1 ;For the first coupling a mixture of 28 7 g of Fmoc-Thr(tBu)-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room 30 temperature 14 1 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasnn resin, and the mixture was agitated for 2 hours ;35 ;Mimosa 15 04 51 ;WO 97/29126 ;- 41 - ;PCT/EP97/00380 ;After filtration, the resin was washed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 ;minutes) and 2 x 500 ml of MeOH (3 minutes) ;5 For the second coupling, a mixture of 14-4 g of Fmoc- ;Thr(tBu)-OH (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved m 400 ml of NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 10 reagent was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc-Thr(tBu)-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)Gln-Nle-Ala-Sasnn resin was washed with 500 ml of 15 DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 24 ;20 Preparation of Fmoc-Tvr<,tBu)-ThrrtBu)-LysfBoc)- ;Leu-ArgCPmc)-LvsCBoo)-Gln-Nle-Ala-Sasnn Resin ;Deprotection of the Fmoc group of Fmoc-Thr(tBu)-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasnn resin was conducted 25 according to the procedure described in Protocol 1 ;For the first coupling a mixture of 24 8 g of Fmoc-Tyr(tBu)-OH (54 mmol), 23 9 of BOP (54 mmol) and 7 3 g of HOBt (54 mmol) was dissolved in 400 ml of NMP with stirring at room 30 temperature 14 1 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Thr(tBu)-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasnn resin, and the mixture was agitated for 2 hours ;35 ;Mimosa 15 04 51 ;WO 97/29126 ^ PCT/EP97/00380 ;After filtration, the resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;5 For the second coupling, a mixture of 12 4 g of Fmoc- ;Tyr(tBu)-OH (27 mmol), 11 9 g of BOP (27 mmol) and 3 65 g of HOBt (27 mmol) was dissolved in 400 ml of NMP and stirred at room temperature 7 05 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 10 reagent was added in one portion to the resin and the mixture was agitated for 2 hours ;After filtration, the Fmoc group of Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasrin resin was 15 washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 ;minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;EXAMPLE 25 ;20 ;Preparation of Fmoc-Asn-TvrftBuVThrftBuVLvsfBoc)-Leu-ArgfPmcVLvsfBocl-Gln-Nle-Ala-Sasrin Resin ;Deprotection of the Fmoc group of Fmoc-Tyr(tBu)-Thr(tBu)-25 Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasrin was conducted according to the procedure described in Protocol 1 before the coupling of Fmoc-Asn-OH via a symmetric anhydride method ;30 19 4 g Fmoc-Asn-OH (54 mmol), and 7 3 g of HOBt (54 ;mmol) were dissolved in a mixture of 135 ml of CH2CI2 and 270 ml of DMF The mixture was stirred in an ice bath before 11 2 g of DCC (54 mmol) was added The mixture was stirred for 30 minutes, was filtered and the filtrate was added in one portion to 35 the Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle- ;Mimosa 15 04 51 ;WO 97/29126 ;- 43 - ;PCT/EP97/00380 ;Ala-Sasnn resin (the (10-18) Sasnn resin) The coupling was completed in 90 minutes ;The resulting resin was then washed with 500 ml of DMF (3 5 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 mlof MeOH (3 minutes) ;For the second coupling, 19 4 g Fmoc-Asn-OH (54 mmol), and 7 3 g of HOBt (54 mmol) were dissolved in a mixture of 135 10 ml of CH2CI2 and 270 ml of DMF The mixture was stirred in an ice bath before 11 2 g of DCC (54 mmol) was added The mixture was stirred for 30 minutes, was filtered and the filtrate was added m one portion to the (10-18)-Sasrin resin The coupling was completed in 90 minutes ;15 ;The resin was then washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) No deprotection was performed after the coupling was completed The final 20 weight of the product, Fmoc-Asn-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Arg(Pmc)-Lys(Boc)-Gln-Nle-Ala-Sasrin resin (Fmoc(9-18)-Sasnn resin), was 112 g ;EXAMPLE 26 ;25 ;Cleavage of Fmoc(9-18) from the Fmoc(9-18)-Sasnn resin ;20g of the Fmoc(9-18)-Sasnn resin was treated with 400 ml of 0 2% TFA m CH2CI2 for 1 minute at room temperature, 30 then filtered The pH of the filtrate was immediately adjusted to pH 7 by the addition of pyridine The filtrate was evaporated, and the residue was triturated with 50 ml of distilled water, then washed with 50 ml of ether The resulting solid material was dried in vacuo ;Mimosa 15 04 51 ;WO 97/29126 ^ PCT/EP97/00380 ;The filtered peptide-resin was then treated six more times with 400 ml of 0 2% TFA solution for 10 minutes followed by adjustment of the pH to 7 with pyridine After evaporation, trituration and drying the HPLC analysis was conducted The 5 conditions for analytical HPLC were column Lichrosorb RP-18, 5 m, 25 cm, eluants (a) 0 1 M HCIO4/H2O (pH 2 5),(b) MeCN, ;gradient 34% to 39% MeCN/20 minutes, flow rate 1 ml/minute, and detector 210 nm Knau All the peptide fragments above 90% purity were combined to give a total of 9 9 g The 52g of product 10 obtained from the cleavage of 112 g of peptide resin had an average purity of 91% ;EXAMPLE 27 ;15 Preparation of Fmoc-GluCOtBuVSasnn Resin ;50 g of 2-metho\y-4-alkoxybenzyl alcohol copolystyrene 1% divinylbenzene cross-linked resin (Sasnn resin) was washed with 500 ml of methylene chloride, and 2 x 500 ml of DMF ;20 ;102 g of Fmoc-Glu(OtBu)-OH (240 mmol) was dissolved in 500 ml of CH2CI2/DMF (9 1 volume ratio) The solution was cooled in an ice bath, then a DCC solution, which was prepared by dissolving 49 5 g of DCC (240 mmol) in 100 ml of CH2CI2/DMF 25 (9 1), was added The mixture was stirred for 30 minutes, then filtered to remove DCU The filtrate was added to the above washed Sasnn resin, followed by the addition of 1 46g of 4-dimethylammopyridine (12 mmol) and 6 27 ml of N-methylmorpholine (60 mmol) The mixture was agitated on an 30 orbital rotary for 9 hours ;The resin was filtered and washed with 500ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 500 ml of MeOH (3 minutes) An aliquot was ;Mimosa 15 04 51 ;WO 97/29126 ;- 45 - ;PCT/EP97/00380 ;removed, dried and the loading was determined by UV analysis to be 0 59 mmol/g ;The resin was washed with 2 x 500 ml of CH2CI2 (3 5 minutes) and 500 ml of DMF (3 minutes) The resin was then suspended in 400 ml of DMF, and 54 3 g of benzoic anhydride (240 mmol) was added, followed by the addition of 100 ml of DMF, and 41 7 ml of dnsopropylethylamine (240 mmol) The suspension was shaken for 30 minutes The Fmoc-Glu(OtBu)-10 Sasrin resin was filtered and washed with 500 ml of CH2CI2 (3 minutes), 500 ml of MeOH (3 minutes), 2 x 500 ml of CH2CI2 (3minutes), 500 ml of DMF (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;15 EXAMPLE 2ft ;Preparation of Fmoc-ThrftBul-GluCOtBuVSasrin Resin ;Deprotection of the Fmoc group of Fmoc-Glu-(OtBu)-Sasrin 20 resin was conducted according to the procedure described in Protocol 1 ;For the first coupling, a mixture of 23 4 g of Fmoc-Thr(tBu)-OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 25 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 15 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Glu(OtBu)-Sasnn resin, and the mixture was agitated for 90 minutes ;30 ;After filtration, the resm was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 , , PCT/EP97/00380 ;- 46 - ;For the second coupling, a mixture of 11 7g of Fmoc-Thr(tBu)-OH (29 5 mmol), 13 1 g of BOP (29 5 mmol) and 4 0 g of HOBt (29 5 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 7 7 ml of DIPEA was added to the above 5 solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes ;After filtration, the Fmoc-Thr(tBu)-Glu(OtBu)-Sasrin resin 10 was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;15 ;EXAMPLE 29 ;Preparation of Fmoc-Phe-Thr(tBu)-GlufOtBu)-Sasnn Resin ;Deprotection of the Fmoc group of Fmoc-Thr(tBu)-Glu(OtBu)-Sasrin resin was conducted according to the procedure 20 described in Protocol 1 ;For the first coupling, a mixture of 22 8 g of Fmoc-Phe-OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 mmol) was dissolved in 400 ml of NMP with stirring at room 25 temperature 15 4 ml 01 DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Thr(tBu)-Glu(OtBu)-Sasrin resin, and the mixture was agitated for 90 minutes ;30 After filtration, the Fmoc-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 An PCT/EP97/00380 ;- 47 - ;The ninhydrin test indicated that the coupling reaction was complete, therefore a second coupling was unnecessary ;EXAMPLE 30 ;5 ;Preparation of Fmoc-Val-Phe-ThrftBuVGlufOtBuVSasrin Resin ;Deprotection of the Fmoc group of Fmoc-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin was conducted according to the procedure 10 described in Protocol 1 ;For the first coupling, a mixture of 20 g of Fmoc-Val-OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 15 15 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin, and the mixture was agitated for 90 minutes ;20 After filtration, the resm was washed with 500 ml of DMF ;(3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;For the second coupling, a mixture of 10 g of Fmoc-Val-OH 25 (29 5 mmol), 13 1 g of BOP (29 5 mmol) and 4 0 g of HOBt (29 5 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 7 7 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was 30 agitated for 90 minutes ;After filtration, the Fmoc-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 35 ml of MeOH (3 minutes) ;Mimosa 15 04 51 ;WO 97/29126 ;- 48 - ;PCT/EP97/00380 ;10 ;30 ;EXAMPLE 31 ;Preparation of Fmoc-Ala-Val-Phe-Thr(tBu)-Glu(OtBu1-Sasrin ;Resin ;Deprotection of the Fmoc group of Fmoc-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin was conducted according to the procedure described in Protocol 1 ;For the first coupling, a mixture of 18 4 g of Fmoc-Ala-OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 15 4 ml of DIPEA was added to the above solution, 15 and the mixture was vigorously stirred The resulting reagent was added in one portion to the Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin, and the mixture was agitated for 90 minutes ;After filtration, the resin was washed with 500 ml of DMF 20 (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;For the second coupling, a mixture of 9 2 g of Fmoc-Ala-OH (29 5 mmol), 13 1 g of BOP (29 5 mmol) and 4 0 g of HOBt (29 5 25 mmol) was dissolved m 400 ml of NMP with stirring at room temperature 7 7 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixtuie was agitated for 90 minutes ;After filtration, the Fmoc-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) ;35 ;Mimosa 15 04 51 ;WO 97/29126 ,n PCT/EP97/00380 ;- 4y - ;EXAMPLE 32 ;Preparation of Finoc-AspfOtBu^-Ala-Val-Phe-ThrftBul-GlufOtBul-Sdsnn Resin ;Deprotection of the Fmoc group of Fmoc-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin was conducted according to the procedure described in Protocol 1 ;10 For the first coupling, a mixture of 24 3 g of Fmoc- ;Asp(OtBu)-OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 15 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting 1*5 reagent was added in one portion, to the Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin, and the mixture was agitated for 90 minutes After filtration, the resin was washed with 500 ml of DMF 20 (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) For the second coupling, a mixture of 12 1 g of Fmoc-Asp(OtBu)-OH (29 5 mmol), 13 1 g of BOP (29 5 mmol) and 40 g 25 of HOBt (29 5 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 7 7 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes 30 After filtration, the Fmoc-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeGH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) 35 Mimosa 15 04 51 WO 97/29126 - 50 - PCT/EP97/00380 EXAMPLE 33 Pieparation of Fmoc-SerftBu)-Asp(OtBu1-Aln- Val-Phe-ThrrtBuVGlufOtBin-Sasnn Resin 5 Deprotection of the Fmoc group of Fmoc-Asp(OtBu)-Ala-Val-Phe-Thr-(tBu)-Glu(OtBu)-Sasrin Resin was conducted according to the procedure described in Protocol 1 10 For the first coupling, a mixture of 22 6 g of Fmoc-Ser(tBu)- OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 15 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent 15 was added in one portion to the Asp(OtBu)-Ala-Val-Phe-Thr- (tBu)-Glu(OtBu)-Sasnn resin, and the mixture was agitated for 90 minutes After filtration, the resin was washed with 500 ml of DMF 20 (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) For the second coupling, a mixture of 11 3 g of Fmoc-Ser(tBu)-OH (29 5 mmol), 13 1 g of BOP (29 5 mmol) and 4 0 g of 25 HOBt (29 5 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 7 7 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes 30 After filtration, the Fmoc-Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) 35 Mimosa 15 04 51 WO 97/29126 - 5i - FCT/EP97/00380 EXAMPLE 34 Preparation of Fmoc-HisfTrtVSerftBuVAspfQtBuVAla-Val-Phe- ThrCtBu)-Glu('OtBu')-Sasrin Resin 5 Deprotection of the Fmoc group of Fmoc-Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin was conducted according to the procedure described in Protocol 1 10 For the first coupling, a mixture of 36 6 g of Fmoc-His(Trt)- OH (59 mmol), 26 1 g of BOP (59 mmol) and 8 0 g of HOBt (59 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 15 4 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent 15 was added in one portion to the Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin, and the mixture was agitated for 90 minutes After filtration, the resin was washed with 500 ml of DMF 20 (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) For the second coupling, a mixture of 18 3 g of Fmoc-His(Trt)-OH (29 5 mmol), 13 1 g of BOP (29 5 mmol) and 4 0 g ol 25 HOBt (29 5 mmol) was dissolved in 400 ml of NMP with stirring at room temperature 7 7 ml of DIPEA was added to the above solution, and the mixture was vigorously stirred The resulting reagent was added in one portion to the resin, and the mixture was agitated for 90 minutes 30 After filtration, the Fmoc-His(Trt)-Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin was washed with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes) 35 Mimosa 15 04 51 WO 97/29126 - 52 - PCT/EP97/00380 EXAMPLE 35 Acetvlation of HisfTrt)-SeraBu)-Asp(QtBu)-Ala-VaI-Phe-ThrCtBu)-G1uCOtBu')-Sasnn Resin 5 The piotected peptide resin Fmoc-His(Trt)-Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasrin resin was treated with 2 x 25% pipendine and washed as described for the deprotection step in Protocol 1 After the deprotection of the 10 Fmoc group, acetylation of the N-terminus amine was conducted as follows A solution of 100 ml of Ac20 and 100 ml of DIPEA in 800 ml of CH2CI2 was added to the peptide resin and the reaction 15 allowed to proceed for 90 minutes The resin was filtered, then washed successively with 500 ml of DMF (3 minutes), 500 ml of MeOH (3 minutes), 500 ml of CH2CI2 (3 minutes) and 2 x 500 ml of MeOH (3 minutes), and 20 dried in vacuo and gave 110 g of protected Ac(l-8)-Sasnn resm, Ac-His(Trt)-Ser(tBu)-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Sasnn resin EXAMPLE 36 25 Cleavage of Ac-HisfTrt)-Ser(tBu)-AspCOtBu)-Ala-Val-Phe-ThrCtBu)-GlufOtBu)-Sasrin Resin 20 g of the protected Ac(l-8) Sasnn resin was treated with 30 400 ml of 0 5% TFA in CH2CI2 for 1 minutes at room temperature, then filtered The pH of the Filtrate was immediately adjusted to pH 7 by the addition of pyridine The filtrate was evaporated, and the residue was triturated with 50 ml of distilled water, then washed with 50 ml of ether The resulting solid material was 35 dried in vacuo The filtered peptide-resin was then treated three Mimosa 15 04 51 WO 97/29126 - 53 - PCT/EP97/00380 more times with 400 ml of 0 5% TFA solution for 10 minutes, followed by adjusting the pH to 7 with pyndine After evaporation, trituration and drying an HPLC analysis was conducted 5 The filtered peptide-resin was treated three more times again with 400 ml of 0 3% TFA solution for 10 minutes, followed by adjusting the pH to 7 with pyridine After evaporation, trituration and drying, HPLC analysis of the peptide fragment was 10 conducted The analytical HPLC conditions were column Lichrosorb RP-18, 5 m, 25 cm, eluants (a) 0 1 M HC104/H20 (pH 2 5), (b) MeCN, gradient 34% to 39% MeCN/20 minutes, flow rate 1 ml/minute, and detector 210 nm Knau 15 All the peptide fragments above 90% purity were combined to give a total of 10 8 g The overall yield obtained from the cleavage of 110 g of peptide resin was 46 g, had an average purity of 95% 20 EXAMPLE 37 Synthesis of Fmoc-Ala-Lvs(Boct-Lvs-TvrftBuVLeu-Asn-Asp-Leu-LysCBocVLvsCBocVGlv-Gly-ThrftBuVNH^fProtected Fmocf 19-31- NH2) 25 Fragment I, Fmoc(26-31)-NH2 (SEQ ID NO 2) (8 91 g, 8 24 mmol) was dissolved in 90 ml of 10% diethylamme in DMF and stirred at room temperature for 2 hours The amine and the solvent were evaporated in vacuo and the residue was triturated 30 with a mixture of hexane and ether (4 1), the solid product collected on a filter and washed with a mixture of hexane and ether (4 1), providing 7 0 g of H-(26-31)-NH2 as a colorless powder (yield 98 9%) Mimosa 15 04 51 WO 97/29126 - 54 - PCT/EP97/00380 Fragment II, Fmoc(19-25)-OH (SEQ ID NO 3) (9 89 g, 8 15 mmol), H-(26-31)-NH2 (7 0 g, 8 15 mmol), HOBt (4 49 g, 29 3 mmol) and DIPEA (6 81 ml, 39 1 mmol) were dissolved m 120 ml of DMF/CH2CI2 (1 1), then stirred in an ice-water bath Solid 5 HBTU (3 71 g, 9 78 mmol) was added portionwise over a 10- minute period to the above solution Stimng was continued at 0° for 30 minutes and then at room temperature for 3 hours The solution was evaporated m vacuo to remove the solvents and the residue was dissolved in CH2CI2 This solution was washed in 10 turn with saturated NaHC03 (3 times), 10% aqueous citnc acid (2 times), water and bnne, and dried over anhydrous MgS04 The solution was filtered and the filtrate was evaporated providing crude protected intermediate Fmoc (19-31)-NH2 as a colorless solid, 17 4 g (yield 100%) Purity was estimated to be about 70% 15 by analytical HPLC EXAMPLE 38 Synthesis of Fmoc-Asn-TvrftBuVThrftBu)-20 Lvs("Boc)-Leu-Arg-(Pmc)-LvsfBoc)-fGln-Nle-Ala-Aia-LvsrBoc)- Lvs-Tvr("tBu)-Leu-Asn-Asp-Leu-Lvs(Boc1-LvsfBoc)-Glv-Glv-ThrftBuVNH?. (Protected FmocC9-31-NH9^ Fmoc(l9-3 1)-NH2 (17 4 g) was dissolved in 90 ml of 10% 25 diethylamine in DMF and stirred at room temperature for 2 hours The amine and the solvent were removed in vacuo, and the residue was triturated with a mixture of hexane and ether (3 1), the solid collected on a filter and washed with a mixture of hexane and ether (3 1), providing 16 67 g of H-(19-31)-NH2 as 30 colorless solid (yield 100%) Fmoc (9-18)-OH (SEQ ID NO 4) (16 59 g, 8 15 mmol), H-(19-31)-NH2 (16 67 g), HOBt (449 g, 29 3 mmol) and DIPEA (6 81 ml, 39 1 mmol) were dissolved in 150 ml of DMF/CH2CI2 (2 1), (the 35 solution was slightly cloudy), then stirred in an ice-water bath Mimosa 15 04 51 WO 97/29126 - 55 - PCT/EP97/00380 HBTU (3 70 g, 9 78 mmol) was added portionwise to the above solution over a 10-rmnute period The solution became clear and stirring was continued at 0° for 30 minutes, and then at room temperature for 3 hours The solution was evaporated in vacuo 5 to remove the solvents and the residue was dissolved in CH2CI2 This solution was washed in turn with saturated NaHCC>3 (3 times), 10% aqueous citric acid (2 times), water and brine and dried over anhydrous MgSC>4 The solution was filtered and the filtrate was evaporated in vacuo to provide 24 77 g crude 10 protected intermediate Fmoc (9-31)-NH2 as colorless solid for a yield of about 79% The product was determined to be about 69% pure by analytical HPLC EXAMPLE 39 15 Synthesis of Ac-HisfTrO-SerftBuVAspCOtBul-Ala-Val-Phe-Thr(tBu)-Glu(OtBu)-Asn-Tvr(tBu)-Thr(tBu)-Thr(tBu)-LvsfBoc)-Leu-Ar{?(Pmc)-Lvs(Boc)-Gln-Nle-Ala-Ala-Lvs(Boc)-Lvs-TvrftBu)-Leu-Asn-Asp-Leu-L vsfBoc VLys(Boc)-Glv-Glv-Thrf tBu)-NHo 20 (Protected Ac(l-31)-NH7^ Fmoc(9-3 1)-NH2 (24 77 g) was dissolved m 100 ml of 10% diethylamine in DMF and stirred at room temperature for 2 hours The amine and the solvent were removed in vacuo, and 25 the residue was triturated with a mixture of hexane and ether (3 1), the solid collected on a filter and washed with a mixture of hexane and ether (3 1), providing 22 56 g of H-(9-31)-NH2 as colorless solid (yield 96 7%) 30 A solution of H-(9-31)-NH2 (22 56 g 6 22 mmol), Ac(l-8)- OH (SEQ ID NO 5) (8 79 g, 6 22 mmol), HOBt (3 43 g, 22 38 mmol), and DIPEA (5 19 ml, 29 84 mmol) in 200 ml of DMF/CH2C12 (11), was stirred in an ice-water bath while HBTU (2 83 g, 7 4o mmol) was added portionwise to the above solution over a 10-minute 35 period The solution was stirred at 0° for 30 minutes and then at Mimosa 15 04 51 WO 97/29126 - 56 - PCT/EP97/00380 room temperature for 3 hours The solution was evaporated and the residue was dissolved in CH2C^2 Thls solutlon was washed m turn with saturated NaHCO-j (3 times), 10% aqueous citric acid (2 times), vater and bnne, and dried over anhydrous MgS04 The 5 solution was filtered and the filtrate was evaporated providing crude protected Ac(l-31)-NH2 as a colorless solid, 29 54 g (yield 72 2%) Purity was estimated to be 78% by analytical HPLC EXAMPLE 40 10 Deprotection of Protected Ac(l-31)-NH-> Synthesis of the cyclic VIP Analog Protected Ac(l-31)-NH2 (29 54 g) was dissolved in 150 ml 15 of a mixture of TFA (135 ml), EDT (4 5 ml), thioanisole (7 5 ml), anisole (3 ml) and stirred at room temperature for 2 hours This solution was evaporated to remove TFA and poured into 500 ml of pre-cooled ether to give a precipitate which was collected and washed thoroughly with ether The product was dried in vacuo 20 to provide 25 5 g of Ac(l-31)-NH2 as a colorless powder having a purity of about 72-75% as estimated by HPLC EXAMPLE 41 25 Purification of the cvchc VIP Analog Purification of the crude peptide Ac(l-31)-NH2 was performed in multiple runs by preparative HPLC on a Delta Prep 3000 system Quantitative HPLC analysis of an aliquot of the 30 crude product of the Zorbax Protection Plus column (vs a standard of the cyclic VIP analog) revealed that 9 95 g of the analog was present in the crude product which weighed 25 5 g For a typical run, the peptide (4 g) was dissolved in 200 ml of 0 1% TFA/H2O and applied to a YMC ODS-A (120A, 15 p) column Mimosa 15 04 51 WO 97/29126 ^7 PCT7EP97/00380 (4 7 x 50 cm) The mobile phase was (A) 0 1% TFA/H2O - (B) 0 1% TFA) 50% MeOH-CH3CN) A gradient elution was run starting at 20% (B) (10 minutes) and then 20% - 50% (B) in 180 minutes at a flow rate of 80 ml/minute UV detection was 5 performed at 215 nm Fractions containing the mam peak were collected and evaluated by analytical HPLC Fractions judged to be of high purity were pooled, concentrated on a cold finger rotary evaporator, and lyophihzed to yield 1 2 g of the cychc VIP analog Ac(l-31)-NH2 The balance of the crude product was 10 processed by the same method to give a total of 8 48 g (85 2% recovery) of purified cyclic VIP analog, overall yield 23 9% Analytical HPLC and capillary electrophoresis confirmed that the product was >99% pure The product was characterized and identity confirmed by amino acid analysis, FAB-MS, optical 15 rotation, ultraviolet absorbance and circular dichroism Mimosa 15 04 51 WO 97/29126 - 58 - PCT/EP97/00380 SEQUENCE LISTING (1) GENERAL INFORMATION 5 (l) APPLICANT (A) NAME F HOFFMANN-LA ROCHE AG (3) STREET Grenzacherstrdsse 124 (C) CITY Basle (D) STATE BS 10 (E) COUNTRY Switzerland (F) POSTAL CODE (ZIP) CH-4010 (G) TELEPHONE 061-6885108 (H) TELEFAX 061-6831395 (I) TELEX 962292/965542 hlr ch 15 (n) TITLE OF INVENTION SYNTHESIS OF VIP ANALOG (in) NUMBER OF SEQUENCES 7 20 (iv) COMPUTER READABLE FORM (A) MEDIUM TYPE Floppy disk (B) COMPUTER Apple Macintosh (C) OPERATING SYSTEM Apple Macintosh (D) SOFTWARE Word 5 1 25 30 (v) CURRENT APPLICATION DATA APPLICATION NUMBER EP (2) INFORMATION FOR SEQ ID NO 1 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 31 amino acids (B) TYPE amino acid (C) STRANDEDNESS single 35 (D) TOPOLOGY linear In) MOLECULE TYPE peptide (ill) HYPOTHETICAL NO 40 (ix) FEATURE (A) NAME/KEY ModiEied-site (B) LOCATION 21 25 (D) OTHER INFORMATION /note= "SIDE CHAIN CYCLIZATION AT 45 LYS21 TO ASP25" (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 50 His Ser Asp Ala Val Phe Thr Glu Asn Tyr Thr Lys Leu Arg Lys Gin 15 10 15 Xaa Ala Ala Lys Lys Tyr Leu Asn Asp Leu Lys Lys Gly Gly Thr 20 25 30 55 Mimosa 15 04 51 WO 97/29126 ^ PCT/EP97/00380 (2) INFORMATION FOR SEQ ID NO 2 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 6 amino acids 5 (B) TYPE ammo acid (C) STRANDEDNESS single (D) TOPOLOGY linear (li) MOLECULE TYPE peptide 10 (ill) HYPOTHETICAL NO 15 (Xl) SEQUENCE DESCRIPTION SEQ ID NO 2 Leu Lys Lys Gly Gly Thr 1 5 20 (2) INFORMATION FOR SEQ ID NO 3 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 7 amino acids (B) TYPE amino acid 25 (C) STRANDEDNESS single (D) TOPOLOGY linear (11) MOLECULE TYPE peptide 30 (ill) HYPOTHETICAL NO (IX) FEATURE (A) NAME/KEY Modi£ied-sice 35 (B) LOCATION 3 7 (D) OTHER INFORMATION /note= "SIDE CHAINS OF LYS3 AND ASP7 ARE CYCLIZED 40 (Xl) SEQUENCE DESCRIPTION SEQ ID NO 3 Ala Lys Lvs Tyr Leu Asn Asp 1 5 45 (2) INFORMATION FOR SEQ ID NO-4 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 10 amino acids (B) TYPE amino acid 50 (C) STRANDEDNESS single (D) TOPOLOGY linear (n) MOLECULE TYPE peptide 55 (ill) HYPOTHETICAL NO Mimosa 15 04 51 WO 97/29126 - 60 - PCT/EP97V00380 (xi) SEQUENCE DESCRIPTION SEQ ID NO 4 Asn Tyr Thr Lys Leu Arg Lys Gin Xaa Ala 5 15 10 (2) INFORMATION FOR SEQ ID NO 5 (i) SEQUENCE CHARACTERISTICS 10 (A) LENGTH 8 ammo acids (B) TYPE amino acid (C) STRANDEDNESS single (D) TOPOLOGY linear 15 (n) MOLECULE TYPE peptide (m) HYPOTHETICAL NO 20 (xi) SEQUENCE DESCRIPTION SEQ ID NO 5 His Ser Asp Ala Val Phe Thr Glu 1 5 25 (2) INFORMATION FOR SEQ ID NO 6 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 13 amino acids (B) TYPE ammo acid 30 (C) STRANDEDNESS single (D) TOPOLOGY lirear (ll) MOLECULE TYPE peptide 15 (ill) HYPOTHETICAL NO (IX) FEATURE (A) NAME/KEY Modifled-site 40 (B) LOCATION 3 7 (D) OTHER INFORMATION /note= "SIDE CHAINS OF LYS3 AND ASP7 ARE CYCLIZED" 45 (xi) SEQUENCE DESCRIPTION SEQ ID NO 6 Ala Lys Lys Tyr Leu Asn Asp Leu Lys Lys Gly Gly Thr 15 10 50 (2) INFORMATION FOR SEQ ID NO 7 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 23 amino acids (B) TYPE amino acid 55 (C) STRANDEDNESS single (D) TOPOLOGY linear Mimosa 15 04 51 </div>

Claims

<div class="application article clearfix printTableText" id="claims">



WO 97/29126 PCT/EP97/00380 

- 61 - 

111) MOLECULE TYPE peptide 

(ill) HYPOTHETICAL NO 

5 

(xi) SEQUENCE DESCRIPTION SEQ ID NO 7 

Asn Tyr Thr Lys Leu Arg Lys Gin Xaa Ala Ala Lys Lys Tyr Leu Asn 10 1 5 10 IS 

Asp Leu Lys Lys Gly Gly Thr 20 

Mimosa 15 04 51 

J * 

62- 

1 O 

Claims 

1 A method foi the synthesis of the compound Ac-(VIP,_31)--NH2 (SEQ ID NO 1) said method compnsing the coupling of Fmoc protected peptide fiagments peptide Fiagment I (SEQ ID NO 2), 

peptide Fragment II (SEQ ID NO 3), peptide Fiagment III (SEQ ID NO 4) and peptide Fiagment IV (SEQ ID NO 5)
2 The method of claim 1, said method comprising 

(a) deprotecting the Fmoc-protecting group of peptide 

Fragment I and coupling the deprotected peptide 

Fragment I with protected peptide Fragment II, 

(b) deprotecting the Fmoc-protecting group of the resulting peptide of step (a) and coupling it with protected 

Fragment III, 

(c) deprotecting the Fmoc-protectmg group of the resulting peptide of step (b) and coupling it with protected 

Fragment IV, 

(d) deprotecting the resulting protected peptide of step (c) 

to yield deprotected Ac-(VIP,_3|)-NH2,
3 The method of claim 2, further compnsing purifying the deprotected peptide Ac-(VIP,.3,)-NH2
4 The method of claim 3, wherein punfication is accomplished via pieparative HPLC
5 A method for the synthesis of a purified compound Ac-(VIP,_3I)-NH2 (SEQ ID NO 1) by coupling four Fmoc protected peptide fragments peptide Fragment I (SEQ ID NO 2), peptide intellectual property offl' of nz 

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and peptide Fragment IV (SEQ ID NO 5), said method compnsing 

(a) deprotecting the Fmoc-protecting group of peptide 

Fragment I, 

(b) coupling the deprotected peptide Fragment I with protected peptide Fragment II yielding protected intermediate Fmoc-(VIPl9.31)-NH2, 

(c) deprotecting the Fmoc-protecting group of intermediate Fmoc -('VIP 19.3|)-NH2 

(d) coupling the deprotected intermediate Fmoc-(VIP,9_3I)-NH2 with protected Fragment III yielding protected intermediate Fmoc-(VIP9.3))-NH2, 

(e) deprotecting the Fmoc-protecting group of intermediate Fmoc-(VIRj 31)-NH2, 

(f) coupling the deprotected intermediate Fmoc-(VIP9.31)-NH with protected Fragment IV yielding protected intermediate Ac-(VIP, 31)-NH2, 

(g) deprotecting the protected peptide Ac-(VIP|.3t)-NH2, and 

(h) purifying the deprotected peptide Ac-(VIP|31)-NH2
6 The method of claim 5, wherein purification in step (h) is accomplished via preparative HPLC
7 The method of claim 5 wherein said Fmoc-protecting group of said peptide fragments and intermediates is deprotected with 10% Et2NH m DMF
8 The method of claim 5 wherein after deprotection, 

fluorene is removed by washing said peptide fragments and intermediates with hexane-ether
9 The method of claim 5, wherein said deprotected peptide fragments and intermediates are intellectual property off!' 

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1 0 JAN 2000 

0 8 s 5 

10 

a) coupled with I 0 equivalent of said protected peptide fragments using HBTU, HOBt in DMF-CH2CI2 using DIPEA, 

(b) evaporated and dissoluted m CH2CI2, and 

(c) extracted with saturated NaHC03 and 10% citric acid
10 The method of claim 9, wherein said coupling is performed at 0°/30 minutes-lhour and 25°/3 hours with 1 2 eq of HBTU, 3 6 eq HOBt and 4 8 eq of DIPEA
11 A peptide selected fiom the gioup consisting of Fmoc-(VIPl9jl)-NH2 (SEQ ID NO 6) and Fmoc-(VIP9.31)-NH2 (SEQ ID NO 7) 

15 12 A protected peptide fiagment selected fiom the gioup consisting of Fmoc-(VIP26_-!1)-NH2 (SEQ ID NO 2), Fmoc-(VIP|9_25)-OH (SEQ ID NO 3), Fmoc-('VIP9.ls)-OH (SEQ ID NO 4), and Ac-(YIP,.8)-OH (SEQ ID NO 5) 

20 13 A peptide fragment selected from the group consisting of the deprotected Fmoc-(VIP2(,_3|)-NH2 (SEQ ID NO 2), the deprotected Fmoc-(VIP,g 25)-OH (SEQ ID NO 3) and the deprotected Fmoc-—(VIP9.ls)-OH (SEQ ID NO 4) 

25 14 Use of the fiagments defined in any one of claims 11 to 13 

foi the preparation of compound Ac-(VIPMI)-NH2 (SEQ ID NO 1) 

15 A method foi the synthesis of the compound Ac-(V1P,.31)-NH2 (SEQ ID NO 1), substantially as hereinbefore 

30 described with paiticular refeience to the foiegoing Examples 37-41 

16 The compound Ac-(VIP, 3I)-NH2 (SEQ ID NO 1), whenever prepared by the method of any one of claims 1 to 10 and 15 

intellectual property "u. 

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